Abstract
Polymerase chain reaction (PCR) is a unique technique in molecular biology and biotechnology for amplifying target DNA strands, and is also considered as a gold standard for the diagnosis of many canine diseases as well as many other infectious diseases. However, PCR still faces many challenges and issues related to its sensitivity, specificity, efficiency, and turnaround time. To address these issues, we described the use of unique ZnO nanoflowers in PCR reaction and an efficient ZnO nanoflower-based PCR (nanoPCR) for the molecular diagnosis of canine vector-borne diseases (CVBDs). A total of 1 mM of an aqueous solution of ZnO nanoflowers incorporated in PCR showed a significant enhancement of the PCR assay with respect to its sensitivity and specificity for the diagnosis of two important CVBDs, Babesia canis vogeli and Hepatozoon canis. Interestingly, it drastically reduced the turnaround time of the PCR assay without compromising the yield of the amplified DNA, which can be of benefit for veterinary practitioners for the improved management of diseases. This can be attributed to the favorable adsorption of ZnO nanoflowers to the DNA and thermal conductivity of ZnO nanoflowers. The unique ZnO nanoflower-assisted nanoPCR greatly improved the yield, purity, and quality of the amplified products, but the mechanism behind these properties and the effects and changes due to the different concentrations of ZnO nanoflowers in the PCR system needs to be further studied.
Highlights
Polymerase chain reaction (PCR), which is one of the most reliable and popular diagnostic techniques, was invented by Kary Mullis back in 1985 [1]
The diffraction peaks were in good conformity with the standard card of zinc oxide (ZnO) powder diffraction file (PDF) #36-1451
The scanning electron microscopy (SEM) images showed that the synthesized ZnO nanoflowers were self-assembled and clearly potrayed the nanopetal-like structure arising from the center of the flowers (Figure 2b)
Summary
Polymerase chain reaction (PCR), which is one of the most reliable and popular diagnostic techniques, was invented by Kary Mullis back in 1985 [1]. PCR technology has its own set of shortcomings and drawbacks, due to which its reliability sometimes can be questionable and debatable. This can be due to certain obstacles and inhibitors such as nonspecific by-products, low yield, and complexity due to guanine and cytosine (GC)-rich nature. PCR inhibitors have a direct effect on the reaction due to their interactions with the nucleic acid and/or their interference with the DNA polymerases or other thermostable enzymes [12]. This type of interaction of binding between to the nucleic acid may alter amplification and can lead to the co-purification of inhibitor and DNA [13].
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